A NOVEL METHOD FOR THE PRODUCTION OF STABILE VACCINES

Abstract

The present invention relates to a method for producing stabilised vaccines, the method comprising: (a) mixing antigens with a solution comprising: (i) chitosan; (ii) at least three different amino acids and/or at least one dipeptide or tripeptide; and (iii) a sugar; and (b) drying the mixture obtained in (a).

Claims

1. A method for producing stabilised vaccines, the method comprising: (a) mixing antigens with a solution comprising: (i) chitosan; (ii) at least three different amino acids and/or at least one dipeptide or tripeptide; and (iii) a sugar; and (b) drying the mixture obtained in (a).

2. The method of claim 1, wherein the at least three amino acids are selected from the groups of (a) amino acids with nonpolar, aliphatic R groups; (b) amino acids with polar, uncharged R groups; (c) amino acids with positively charged R groups; (d) amino acids with negatively charged R groups; and (e) amino acids with aromatic R groups.

3. The method of claim 1, wherein the solution comprises at least one amino acid selected from each group of (a) an amino acid with nonpolar, aliphatic R groups; (b) an amino acid with polar, uncharged R groups; (c) an amino acid with positively charged R groups; (d) an amino acid with negatively charged R groups; and (e) an amino acid with aromatic R groups.

4. The method according to claim 1, wherein the solution comprises at least the amino acids selected from: (a) alanine, glutamate, lysine, threonine and tryptophane; (b) aspartate, arginine, phenylalanine, serine and valine; (c) proline, serine, asparagine, aspartate, threonine, phenylalanine; (d) tyrosine, isoleucine, leucine, threonine, valine; (e) arginine, glycine, histidin, alanine, glutamate, lysine, tryptophane; and (f) alanine, arginine, glycine, glutamate, lysine.

5. The method according to claim 1, wherein one or more of the amino acids are selected from natural non-proteinogenic amino acids and synthetic amino acids.

6. The method according to claim 1, wherein at least one of the dipeptide(s) in accordance with claim 1(a)(ii) is selected from carnosin, glycyltyrosine, glycylglycine and glycylglutamine.

7. The method according to claim 1, wherein the sugar is trehalose.

8. The method according to claim 1, wherein the solution further comprises at least one saponine.

9. The method according to claim 1, wherein the antigens are split virus antigens.

10. The method according to claim 1, wherein the split virus antigens are influenza virus antigens.

11. The method according to claim 10, wherein the influenza virus is an influenza A virus.

12. The method according to claim 10 or 11, wherein the influenza virus is an influenza A H1N1 virus.

13. The method according to claim 1, wherein the step of drying the mixture is achieved by a method selected from spray drying, lyophilisation, spray-freeze drying and air drying.

14. The method according to claim 1, wherein the dried vaccine obtained in step (b) is subsequently sterilized.

15. The method according to claim 1, wherein the vaccine is for intramuscular, subcutaneous, intradermal, transdermal, oral, peroral, nasal, and/or inhalative application.

Description

[0114] The figures show:

[0115] FIG. 1: Differential Scanning Fluorimetry

[0116] Normalized thermal denaturation curves of the model protein in combination with different stabilizing excipients alone or excipient mixtures compared to the corresponding thermal denaturation profile of the model protein in PBS buffer. Thermal denaturation curves of the model protein in different concentrations of trehalose (A), chitosan (B), SPS (C), SPS+trehalose (D), SPS+chitosan (E) and SPS+trehalose+chitosan (F), Comparison of the normalized thermal denaturation curves of the analysed model protein for all excipients and excipient mixture in a selected concentration range (G).

[0117] FIG. 2: SDS-PAGE monitoring of the selected variants over time.

[0118] Non-reducing SDS-PAGE (A) and reducing SDS-PAGE (B) of the different formulations of the influenza vaccine before and after spray-drying and subsequent irradiation at 25 kGy (e-beam) at the time point t=0. The samples are loaded for each treatment condition in the following order: Mark12 (lane 1); liquid SPS T final prior to SD (lane 2); SD SPS Trehalose final (lane 3); SD SPS T final 25 kGy (lane 4); original Pandemrix (positive control; lane 5); liquid M prior to SD (lane 6); SD M 25 kGy (lane 7); Mark12 (lane 8). Non-reducing SDS-PAGE (C and E) and reducing SDS-PAGE (D and F) of the different formulations of the influenza vaccine before and after spray-drying and subsequent irradiation at 25 kGy (e-beam) at the time point t=1 month (C, D) and t=3 months (E, F). The samples are loaded for each treatment condition in the following order: Mark12 (lane 1); SD SPS T final 2-8 C. (lane 2); SD SPS T final 25 C. 60% rH (lane 3); SD SPS T final 25 kGy 2-8 C. (lane 4); SD SPS T final 25 kGy 25 C. 60% rH (lane 5); SD M 25 kGy 2-8 C. (lane 6); SD M 25 kGy 25 C. 60% rH (lane 7); original Pandemrix+chitosan-HCl (lane 8); original Pandemrix (positive control; lane 9); Mark12 (lane 10).

[0119] FIG. 3: Immune responses in macaques to Pandemrix vaccine. A, Haemagglutination inhibition assay (HAI) titre; B, Microneutralisation assay (MN) titre. In each chart, bars represent the geometric mean titre for each group. Group 1: negative control group (PBS); Group 2: original Pandemrix; Group 3: SD M 25 kGy; Group 4: SD SPS T final; Group 5: SD SPS T final 25 kGy. *, significant difference from group 2, 4 and 5 means, p<0.01. Means for groups 2, 4 and 5 are not significantly different from one another at any time-point.

[0120] FIG. 4: Dynamic light scattering. Particle size distribution by dynamic light scattering of A) original Pandemrix; SD M; SD M 25 kGy; SD SPS T final; SD SPS T final 25 kGy B) SD M 25 kGy, prepared freshly and after 1 and 3 month storage at 2-8 C. and 25 C., 60% rH C) SD SPS T final; prepared freshly and after 1 and 3 month storage at 2-8 C. and 25 C., 60% rH D) SD SPS T final 25 kGy, prepared freshly and after 1 and 3 month storage at 2-8 C. and 25 C., 60% rH

[0121] FIG. 5: SEC-Analyses. SEC chromatograms of A) original Pandemrix; SD M; SD M 25 kGy; SD SPS T final; SD SPS T final 25 kGy B) SD M 25 kGy, prepared freshly and after 1 and 3 month storage at 2-8 C. and 25 C., 60% rH C) SD SPS T final; prepared freshly and after 1 and 3 month storage at 2-8 C. and 25 C., 60% rH D) SD SPS T final 25 kGy, prepared freshly and after 1 and 3 month storage at 2-8 C. and 25 C., 60% rH.

[0122] FIG. 6: Water content in % of different formulations after spray drying as determined by Karl-Fischer titration. Results are shown as meanSD values (n=3).

[0123] FIG. 7: LDH enzyme activities normalized in % of a standard activity in 4 SPS variants upon liquid storage at a temperature of 50 C. before storage t=0 days and after 2 days, 4 days, 7 days and 14 days in comparison to the storage of LDH in 100 mM sodium phosphate buffer. The measured LDH activity was normalized to a standard curve.

[0124] FIG. 8: Comparison of the specific LDH enzyme activity before (t=0 days) and after liquid storage at the indicated time points (t=14; 16; 18 and 21 days) formulated with either trehalose and chitosan (left side of the figure) or with a mixture of 7 amino acids, trehalose and chitosan (right side of the figure).

[0125] FIG. 9: LDH enzyme activities normalized in % of a standard activity before (t=0) and after liquid storage of LDH formulated with the main components employed in WO 2009/014774 (Pluronic F127 and rHSA) as compared to a combination of 7 amino acids, trehalose and chitosan with these components at 50 C. measured at the indicated time points (t=2; 4 and 7 days).

[0126] The examples illustrate the invention.

EXAMPLE 1: MATERIALS AND METHODS

Stabilizing and Protecting Solution (SPS)

[0127] The proprietary aqueous Stabilizing and Protecting Solution (SPS; LEUKOCARE, Munich, Germany) is composed of different small molecules (here mostly amino acids) and glycosidic excipients (here, glycyrrhizinic acid) usually provided as a stock concentration of 80 mg/mL (pH 7). All components are used in pharmaceutical quality; they are non-toxic and routinely used in parenteral solutions.

Influenza Strain

[0128] Pandemrix (Glaxo Smith Kline) was used as the antigen vaccine. Pandemrix is provided in two vials, one of which contains an influenza (split virion, inactivated) A/California/7/2009 (H1N1)v like strain (x-179a) that comprises the main antigen hemagglutinin. The second vial comprises the adjuvant, AS03 (squalene (10.69 mg per dose), DL--tocopherol (11.86 mg) and polysorbate 80 (4.86 mg)). 15 g/ml of the antigen dispersion (are usually mixed 1:1 with adjuvant prior to injection (3.74 g HA per dose 500 l). For the present study, the split viral antigens of the first vial were re-formulated as described, dried and sterilised. Prior to injection, said re-formulated antigen preparations were resuspended and mixed 1:1 with adjuvant, as described above.

Differential Scanning Fluorimetry

[0129] SYPRO orange (5000 stock solution; Life Technologies (Carlsbad, Calif.) was diluted 1:1000 in different concentrations of the stabilizing excipient mixtures or in PBS buffer to a 5 final concentration. In the next step the model protein was diluted to 300 g/ml and 600 g/ml, respectively with the 5SYPRO orange solutions of these different concentrations of the stabilizing excipients or with buffer and subsequently distributed as 50 l aliquots into the wells of a 96-well PCR plate (4titude, Berlin). The PCR plates were sealed with a PCR film (4titude, Berlin) and 1 min centrifuged at 500 g at room temperature to avoid creating air bubbles and to collect the solution at the bottom of the wells. The plates were subsequently heated on a q-PCR Light-Cycler 480 II (Roche) from 20 to 95 C., with a ramping rate of 3 C. min.sup.1. The set up of the filter configuration was the optimal excitation wavelength of 498 nm and emission wavelength of 610 nm for SYPRO orange. The midpoint of thermal denaturation T.sub.m was calculated by fitting the data to the Boltzmann equation using GraphPad Prism 6. The differences in T.sub.m between the control samples of the model protein in PBS and the stabilizing excipients containing formulations were calculated as thermal shift.

Formulation Variants

[0130] For in vitro testing, a range of formulation variants were spray dried of which a small number was selected for the in vivo study. A summary of the formulation variants and names used throughout the present application are given in Table 3. Original Pandemrix was either used as supplied and mixed with the matrix component in the respective amount or in case of the SPS containing formulations it was dialysed overnight against SPS in the respective concentrations at pH 7, using Slide-A-Lyzer dialysis cassettes (cut-off 3.5 kDa; volume 3-12 mL) (Thermo Scientific, Schwerte-Geisecke, Germany). The additional components mannitol, trehalose and glycyrrhizinic acid (Sigma-Aldrich, Munich, Germany), respectively, were added afterwards. For in vivo batches, chitosan-HCl was also added (Heppe Medical Chitosan GmbH, Halle a. d. Saale, Germany).

Spray-Drying

[0131] A Bchi B-290 laboratory spray-dryer (Bchi, Flawil, Switzerland) was used for spray-drying of the vaccine preparations (see table 4 for parameters). The dried product was collected in the product vessel using a high performance cyclone, sealed in glass vials and was stored at 2 to 8 C. until analysis. For the batches of the in vivo study, all components of the spray-dryer were disinfected with 70% (V/V) isopropanol prior to use. Dried products were filled in individual doses in pre-sterilised glass vials (Type I, 2R, PRI-PAC e.K. Eschweiler, Germany) under aseptic conditions.

Haemagglutination Assay

[0132] Dried samples were reconstituted in water (15 g HA/mL). To determine HA titres, 50 L of the diluted split vaccine formulations were two-fold serially diluted in 50 L PBS mixed with an equal volume of fresh 0.5% (W/V) chicken red blood cell suspension (Harlan Laboratories, Belton, Leicestershire, UK) in a U-bottom 96 well microtitre plate. After one hour incubation at room temperature, the plates were scored for agglutination. The titres are given as inverse of the highest dilution causing the agglutination of red blood cells.

Electrophoresis

[0133] Spray dried products were reconstituted in water (15 g HA/mL). For denaturing conditions, the samples were prepared by mixing 12 L of the vaccine formulation with 12 L of the NuPAGE LDS-sample buffer 2 concentrate (Invitrogen, Darmstadt, Germany). Separation was performed at a constant voltage of 200 V and running time was 90 min. Gels were stained with a silver staining kit (SilverXpress silver staining kit, Invitrogen). A molecular weight standard (Novex Mark 12 Unstained Standard, Invitrogen) was analysed on each gel.

Irradiation Protocol

[0134] Sterilisation of the spray dried and freeze dried vaccine samples was performed in sealed glass vials by BGS, Saal a. d. Donau, Germany, using -irradiation at 25 or 40 kGy. Samples for the in vivo study and long term storage were -irradiated with 25 kGy.

Long Term Storage

[0135] For the formulations chosen for the in vivo study, extensive characterisation directly after production and over storage was performed. All formulations were packed in glass vials, were sealed and stored at 2 to 8 C., or at 25 C. (+/2 C.) at 60% (+/5%) relative humidity (rH), respectively. After one month and after three months, samples were analysed. Characterisation included haemagglutination assay, DLS measurements, water content, SEC profiling and electrophoresis.

Size Exclusion Chromatography (SEC)

[0136] Size exclusion chromatography (SEC) was performed by using an Merck-Hitachi D 7000 system (Merck-Hitachi, Darmstadt, Germany) with UV detector at 214 nm and a 13 m TSKgel GMPWXL SEC column (7.8300 mm) (TOSOH Bioscience GmbH, Stuttgart, Germany). Samples were run in a mobile phase of PBS pH 7.4 (flow rate 0.7 mL/min). A sample amount equivalent to 15 g HA was used for each analysis. All measurements were performed in duplicates.

Dynamic Light Scattering (DLS)

[0137] Dynamic Light Scattering was performed to analyse for protein aggregates using a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK). A sample amount equivalent to 15 g of protein (HA) was redispersed in double distilled water and was measured. All results are mean of three sets of 30 individual scans.

Water Content

[0138] 50 mg of the spray dried product was dissolved in 1 mL of dimethylsulfoxide (DMSO) and added to the titration flask of the Karl Fischer titrator (V20, Mettler Toledo AG, Schwerzenbach, Switzerland). Titration was then carried out using Karl Fischer reagent of previously determined titre (mg H.sub.2O/mL). Water content was determined in triplicate for each sample and was subtracted for solvent. Results are shown as meanSD values.

Animal Study

[0139] The study was approved by the Ethical Review Process of Public Health England, Porton, Salisbury, UK and the Home Office via Project License PPL30/2993. The study was conducted in accordance with the PHE Porton Down Quality Management System that is compliant with BS EN ISO9001-2000.

[0140] Animals (Macaca fascicularis) consisted of mature adults with equal distribution of male and female animals in each group. Twenty-six mature adult animals of either sex (age range 4 to 6 years and weight range 3.6 to 5.9 kg at the start of the study) were obtained from a Home Office accredited breeding colony within the United Kingdom. All animals were maintained within a conventional colony tested to be free of Herpesvirus simiae (B-virus), Mycobacterium tuberculosis (TB), Simian T-cell Lymphotropic virus (STLV) and Simian immunodeficiency virus (SIV) and were selected from a cohort of animals screened for the absence of influenza antibodies.

[0141] The animals were housed in their existing social groups in pens designed in accordance with the requirements of the United Kingdom Home Office Code of Practice for the Housing and Care of Animals Used on Scientific Procedures (1989). Each animal was individually identified by a permanent tattoo using a unique number. Tap water and Expanded Primate Maintenance diet (PME, Special Diet Services, UK) were available ad libitum with enrichment treats, vegetables and fruit provided on a regular basis.

TABLE-US-00001 Animal Groups: Group 1: Negative control (PBS) 2 animals Group 2: Original Pandemrix 6 animals Group 3: SD M 25 kGy 6 animals Group 4: SD SPS T final 6 animals Group 5: SD SPS T final 25 kGy 6 animals

[0142] Original Pandemrix consisted of the human vaccine formulation, including the AS03 adjuvant provided as a separate flask. The spray dried products were supplied in single dose vials and were reconstituted with sterile water and AS03 adjuvant immediately prior to vaccination. Each animal was given 0.5 ml vaccine preparation containing 3.75 g HA antigen (human adult dose). All vaccinations were given by intramuscular injection. Control sera were taken eight days prior to vaccination, and sera were taken at 21, 34 and 48 days post-vaccination. A booster immunisation was given 28 days post-vaccination, with an equal dose of the appropriately-treated vaccine. At every sampling or vaccination occasion each animal was weighed, had a rectal temperature taken, superficial lymph nodes (inguinal and axillary) palpated, the site of vaccination examined and a check was made of general physical condition. In addition, haemoglobin levels were checked at each blood sampling occasion.

Haemagglutination Inhibition Assay (HAI)

[0143] Sera were treated with receptor-destroying enzyme (RDE, Denka Seiken Co., Japan), followed by heat-inactivation. Treated sera were then subjected to 2-fold serial dilutions in 96-well U-bottom plates, followed by the addition of 4 HA units of virus (influenza A/California/07/09). After incubating at room temperature, a 0.5% w/v suspension of chicken red blood cells was added as described above for the HA assay. The end-point was defined as the highest serum dilution showing complete HA inactivating activity.

Microneutralisation Assay (MN)

[0144] RDE-treated sera were subjected to 2-fold serial dilutions in 96-well cell culture plates. The diluted sera were mixed with an equal volume of medium containing 100 TCID50 influenza A/California/07/09 virus. After incubation, 100 l of Madin-Darby canine kidney (MDCK) cells were added to each well. The plates were incubated for 18 to 20 hours. Cell monolayers were washed with PBS and fixed. Presence of viral antigen was detected with a primary antibody to the influenza A NP protein followed by a secondary peroxidase conjugate. After staining, absorbance was read at 492 nm. The reciprocal serum dilution corresponding to the lowest dilution to be scored negative for neutralising activity is the 50% neutralisation antibody titre.

Data Analysis.

[0145] Data from animal study are depicted as geometric mean values of n=6 animals per group. Minitab 15 software was used to conduct nonparametric analyses using Mann-Whitney Rank Sum Test. Intergroup differences were considered significant at p<0.01.

EXAMPLE 2: DIFFERENTIAL SCANNING FLUORIMETRY (DSF)THERMAL SHIFT ASSAY

[0146] Thermal profiles of a model protein were monitored in the presence of an environmentally sensitive fluorescent dye which is highly fluorescent in non-polar environment, such as the hydrophobic sites of unfolded proteins, compared to the aqueous solution where the fluorescence is quenched. The temperature at which a protein unfolds is measured by an increase in the fluorescence of the applied dye with affinity to hydrophobic parts of the protein, which are exposed as the protein unfolds. The plot of the fluorescence intensity as a function of temperature generated a sigmoidal curve that can be described by a two state transition. The inflection point of these transition curves (T.sub.m) was calculated by fitting the curves using a Boltzmann equation.

[0147] A T.sub.m 70.2 C. and 70.7 C. dependent of the protein concentration was obtained for the model protein in PBS Buffer. Addition of trehalose alone to the protein solution resulted in a thermal shift between 2 to 3 C. with a slight decrease in the higher concentration range of trehalose (FIG. 1A, G and Table 1). In contrast, already the addition of chitosan alone led to a more remarkable stabilizing effect on the thermal denaturation profile of the model protein. The thermal shift of the T.sub.m with chitosan was between 3 to 5 C. with an increase with increasing chitosan concentrations (FIG. 1B, G and table 1). The thermal shift of the model protein between 2.5 and 3.2 C. with an excipient mixture based on the SPS platform technology was approximately comparable with the stabilizing effect of trehalose in this assay (FIG. 1C, G and table 1). After addition of chitosan to this excipient mixture based on the SPS platform technology, a pronounced concentration-dependent increase of the thermal shift was found. Greater than 3 C. in the small concentration range to approximately 9 C. in the higher concentration range was determined for this excipient mixture (FIG. 1E, G and table 1). In the case of the addition of trehalose to the SPS based excipient mixture a concentration dependent increase of the thermal shift from 2.5 C. to 6.5 C. was found (FIG. 1D, G and table 1). Further addition of chitosan to the mixture of SPS and trehalose led to additional enhanced thermal shifts in a concentration dependent manner. The T.sub.m of the model protein increases with increasing concentrations of the mixture from 2.7 to nearly 11 C., suggesting a synergistic stabilizing effect of chitosan in combination with the different excipient mixtures (FIG. 1F, G and table 1). For the further study considering the stability of the H1N1 split vaccine, the mixture of chitosan, SPS and trehalose with the highest concentration and the corresponding highest thermal shift of 11 C. was applied.

EXAMPLE 3: STRUCTURAL ANALYSIS

[0148] To correlate the functional activity of the SPS-formulated vaccines with the retention of structural integrity, SDS-PAGE migration patterns of these samples were compared with the positive control (FIG. 2). Non-reducing and reducing SDS-PAGE analysis of original Pandemrix indicated typical migration patterns for highly purified split vaccines. Non-reducing SDS-PAGE showed six separated bands in the case of the original Pandemrix (FIG. 2A).

[0149] Because of the highly hydrophobic nature of the integral membrane protein haemagglutinin and the resulting high susceptibility to form soluble aggregates in solution and protein complexes with the other protein components, an assignment of the single bands to single components is very difficult. The lack of the protein bands migrating at molecular weights of 200 kDa and between 200 and 116.5 kDa under reducing conditions indicated that the oligomeric forms observed under non-reducing conditions were disulphide linked oligomers of HA0 particularly dimers and trimers (FIGS. 2A and B). The bands at approximately 65 kDa in the non-reducing SDS-PAGE may correspond to the likewise disulphide linked HA0 monomers, not visualised by reducing SDS-PAGE. The band at approximately 55.4 kDa could correspond to smaller amounts of HA1 in complex with the nucleoprotein. A small band between 31 and 21.5 kDa may correspond to the protein complex of HA2 and the matrix protein M1. The small amount of degradation of the disulphide-linked HA0 and the dimer and trimer, respectively could be a consequence of the sample preparation for the non-reducing gelheating of the samples for 10 min at 90 C. leading to maximum binding of SDS to the protein (FIG. 2A). Upon the loss of the bands corresponding to the disulphide-linked HA0 monomer and the haemagglutinin dimer and trimer two prominent bands in the reducing SDS-PAGE may correspond to the HA1 and HA2 subunits of haemagglutinin in complex with nucleoprotein and matrix protein, respectively (FIG. 2B).

[0150] It is known from literature that nucleoproteins and matrix proteins, being present in the split vaccine formulation, can interfere with the detection of haemagglutinin [26, 31]. In the case of the liquid SD M before spray-drying the same migration pattern as the positive control in the non-reducing as well as in the reducing SDS-PAGE was found (FIGS. 2A and B). In contrast, SD M showed a considerable loss of band intensity corresponding to the main antigenic components of the split vaccine (data not shown), matching the pronounced loss of functional integrity in the HA-assay. Subsequent -irradiation of this formulation at 25 kGy led to almost complete loss of the typical migration pattern, suggesting substantial fragmentation of the protein constituents (FIGS. 2A and B). From the migration patterns of the SPS formulated vaccines it can be seen that the chitosan-containing preparations liquid SPS T final (before SD), SD SPS T final and SD SPS T final 25 kGy (FIGS. 2A and B, lanes 2 to 4) resulted in a long smear overlaying individual bands. It is well-known that chitosan can bind to lipids [29]. Thus, chitosan-HCl might bind to the residual fragments of the lipid membrane of the split vaccine in these formulations, which may cause this effect. There was no loss of any protein band and particularly of haemagglutinin in SPS protected vaccines after spray-drying and irradiation.

EXAMPLE 4: ANALYSIS OF STORAGE STABILITY

SDS PAGE Electrophoresis

[0151] After storage times of one month (FIGS. 2C and D; lanes 2 to 5) and three months (FIGS. 2E and F; lanes 2 to 5) at 2 to 8 C. and at 25 C. (rH 60%), the analysis showed no changes in the migration pattern of the SD SPS T final and SD SPS T final 25 kGy compared to the corresponding migration pattern at t=0 (FIGS. 2A and B; lanes 2 to 4). In the case of SD M 25 kGy, storage for 1 and 3 months at 25 C. (rH 60%) led to a further loss of residual protein components of the split vaccine (FIGS. 2C, D, E, and F, lane 7) compared to storage at 2 to 8 C. (FIGS. 2C, D, E, and F; lane 6) and time point t=0 (FIGS. 2A and B; lane 7). This shows that the SPS-stabilised spray dried and particularly the irradiated formulation remained stable not only upon refrigerated storage, but also at 25 C., 60% rH demonstrating increased thermo stability.

Dynamic Light Scattering (DLS)

[0152] DLS data displays the size of colloidal components and aggregates. As visible in FIG. 4a, original Pandemrix consists of colloidal components of a certain size distribution, but no large aggregates were visible. After spray drying, aggregates were visible for the SD M formulation as indicated by the second peak at around 5000 nm, but not for the SD SPS T final formulation (FIGS. 4a and b). After irradiation a slight increase in aggregation can be seen for the SD SPS T final 25 kGy formulation (FIGS. 4a and d). On storage for one month and three months, respectively, the spray dried and the subsequently irradiated formulations with SPS did not show an increase in aggregation irrespective of the storage conditions as shown in FIGS. 4c and d. This indicates that no destabilising processes take place over storage time which would increase aggregation upon redispersion.

Size Exclusion Chromatography

[0153] In size exclusion chromatography the highly hydrophobic nature of the protein led to elution as protein complexes. SEC chromatograms are shown in FIG. 5. The first peak at about 11 ml in the original Pandemrix could be assigned to a high molecular weight complex of the protein constituents>500 kDa. The second peak at 16 ml may represent the HA trimer of 255 kDa; it became larger in the SD M and is heavily increased in the SD M 25 kGy (FIG. 5a). The third peak could be the HA monomer, which is totally lost in the corresponding irradiated formulation SD M 25 kGy (FIG. 5a). The SD SPS T final exhibits the same peaks of the trimer and the monomer, but lost the high molecular weight complex (FIGS. 5a and c) indicating a stable formulation.

[0154] In contrast, SD M 25 kGy showed changes in the trimer peak area, especially upon storage at 25 C./60% rH for 1 and 3 months (FIGS. 5a and b) indicating that the antigen was not stabilised properly leading to increased aggregation upon redispersion. SEC chromatographs (FIGS. 5c and d) showed no change in the SD SPS T final or the SD SPS T final 25 kGy formulation after storage for 1 and 3 months at either storage condition compared to the freshly prepared formulation.

Water Content

[0155] As storage stability was performed under tightly sealed conditions, a remarkable change would indicate an improper sealing. No such effect was observed (FIG. 6).

EXAMPLE 5: IN VIVO ANALYSIS

[0156] Throughout the experimental period all animals maintained weight, haemoglobin level and body temperature within the expected range for the species. No adverse reactions were observed at the site of injection and no lymphadenopathy was detected by palpation. 21 days post-vaccination, all animals in groups 4 and 5, and five out of six animals in group 2, had sero-converted as shown by an HAI titre40 (FIG. 3a). In contrast, all six animals in group 3 (SD M 25 kGy) remained sero-negative (HAI titres20). These observations were confirmed by MN titres which showed group 3 sera to be equivalent to mock-vaccinated control sera (MN titres 40-80), with all animals in groups 2, 4 and 5 showing MN titres of between 160 and 5120 (FIG. 3b).

[0157] All animals then received a booster vaccination 28 days after primary vaccination, and sera were taken for analysis 6 and 20 days post-boost. Following boost, all animals in group 3 showed seroconversion by HAI and MN titres. However, mean titres for group 3 remained significantly lower than mean titres of groups 2, 4 and 5 in both assays (FIG. 3). Mean titres of groups 2, 4 and 5 were not significantly different from one another. Mock-vaccinated animals were boosted with PBS and did not sero-convert. These data demonstrate that spray-drying and irradiation led to >10-fold reduction in mean HAI titre and >15-fold reduction in mean MN titre in the absence of protection; whereas in the presence of the inventive solution, there was no significant reduction in titre due to irradiation. Furthermore, spray-drying of vaccine in the presence of the inventive solution had no detrimental effect on immunogenicity compared to untreated vaccine.

EXAMPLE 6: STORAGE STABILITY OF THE MODEL ENZYME LACTIC DEHYDROGENASE (LDH) AT 50 C. IN THE LIQUID STATE

[0158] Further experiments were carried out to show the influence of individual compounds on the stability of proteins during storage. In addition, a direct comparison with the results described in WO 2009/014774 was carried out, where a combination of chitosan with trehalose, Pluronic F127 and rHSA was allegedly shown to decrease the antigen titre loss upon incubation at 37 C. However, the presence of rHSA in the samples renders it impossible to analyse the effect on the above employed split virus preparation, as the rHSA would present in an SDS-PAGE analysis at the same band size as hemagglutinin. To nonetheless enable a direct comparison, the following experiments were carried out using the model enzyme lactic dehydrogenase (LDH) as proof of concept. LDH is a commonly employed model protein employed in the development of spray dried formulations, freeze drying and the effect of storage on this enzyme can conveniently be analysed in enzyme assay readouts.

Materials and Methods

[0159] Lactic dehydrogenase (LDH; Sigma-Aldrich, Munich, Germany) was recombinantly expressed in E. coli and was used as the model protein, for the reasons detailed above. A 50 mg/ml stock solution of LDH was prepared by dissolving the lyophilized powder of the enzyme in 10 mM sodium phosphate buffer pH 7.5. For the preparation of the formulation variants the LDH stock solution was further diluted to an enzyme concentration of 1 mg/ml with the respective formulations. The LDH formulations were subsequently incubated at 50 C. for 21 days and the enzyme activity of lactic dehydrogenase was measured at the indicated time points (t=0; 2; 4; 7; 14; 16; 18; and 21 days).

[0160] LDH enzymatic activity was determined by monitoring the decrease in absorbance of the reduced cofactor NADH at a wavelength of 340 nm and at a temperature of 22 C. upon the enzymatic reaction of pyruvate to lactate. Before measuring the activity, the formulations were diluted to a concentration of 37.5 g/ml with 100 mM sodium phosphate buffer pH 7.5. A reaction mixture of 790 l sodium phosphate buffer pH 7.5; 100 l pyruvate stock solution (20 mM) and 100 l NADH stock solution (1 mM) was prepared and the enzymatic reaction was started by addition of 10 l LDH dilution (37.5 g/ml).

Results

[0161] The enzymatic activity of LDH after liquid storage at 50 C. was measured at various time points. FIG. 7 summarises the results obtained for LDH storage after formulation with one of 4 different SPS variants in comparison to a buffer formulation. SPS 17 is a mixture of 4 amino acids, SPS 18 is a mixture of 4 amino acids with trehalose, SPS 19 is a mixture of 4 amino acids in combination with chitosan and SPS 20 is a mixture of 4 amino acids in combination with trehalose and chitosan.

[0162] As is shown in FIG. 7, when employing only a buffer formulation, there was a significant loss of activity upon storage at 50 C. In contrast, employing any of the 4 SPS variants protected LDH from such a loss of activity. Moreover, the addition of chitosan in both cases (see SPS 19 and SPS 20) increased the enzymatic activity of LDH upon liquid storage compared to the same SPS formulations without chitosan (SPS 17 and SPS 18).

[0163] FIG. 8, left group of bars, shows that a mixture of trehalose and chitosan alone, without amino acids, resulted in a dramatic loss function of the enzyme after liquid storage at 50 C. for 16; 18 and 21 days. The addition of amino acids to this mixture of trehalose and chitosan, however, was capable of leading to the retention of the enzymatic function during liquid storage of LDH at 50 C. up to 21 days. For a direct comparison with the results described in WO 2009/014774, the main components of WO 2009/014774, namely Pluronic F127 and rHSA, were combined with either trehalose, or with trehalose and chitosan, or with amino acids, trehalose and chitosan. As is shown in FIG. 9, right group of bars, the mixture of amino acids, trehalose and chitosan with Pluronic F127 and HSA led to an increase of the LDH activity between 20 and 40% after storage at 50 C. as compared to a mixture of Pluronic F127 and HSA with either trehalose alone (left group of bars) or with trehalose and chitosan (middle group of bars).

Tables:

[0164]

TABLE-US-00002 TABLE 1 Calculated midpoints of thermal denaturation T.sub.m from the thermal denaturation plots corresponding to the model protein 300 g/ml in buffer and in the analysed excipient mixtures in different concentrations (order C1 to C8 with increasing concentration). C 1 C 2 C 3 C 4 C 5 C 6 C 7 C 8 T.sub.m T.sub.m T.sub.m T.sub.m T.sub.m T.sub.m T.sub.m T.sub.m Formulation [ C.] [ C.] [ C.] [ C.] [ C.] [ C.] [ C.] [ C.] PBS 70.7 70.7 70.7 70.7 70.7 70.7 70.7 70.7 trehalose 73.7 73.2 73.7 73.5 71.9 72.7 72.8 72.8 chitosan 73.9 74.0 73.9 74.0 74.2 74.9 75.9 75.4 SPS 73.2 73.1 n.d. 73.7 73.5 73.5 73.3 73.8 SPS + 72.3 n.d. 73.0 74.4 75.7 76.6 n.d. 76.0 trehalose SPS + 73.6 73.9 73.9 74.3 75.6 77.5 n.d. 79.3 chitosan SPS + 73.4 73.7 73.2 74.2 76.0 78.4 80.9 81.5 trehalose + chitosan

TABLE-US-00003 TABLE 2 Calculated midpoints of thermal denaturation T.sub.m from the thermal denaturation plots corresponding to the model protein 600 g/ml in buffer and in the analysed excipient mixtures in different concentrations (order C1 to C8 with increasing concentration). C 1 C 2 C 3 C 4 C 5 C 6 C 7 C8 T.sub.m T.sub.m T.sub.m T.sub.m T.sub.m T.sub.m T.sub.m T.sub.m Formulation [ C.] [ C.] [ C.] [ C.] [ C.] [ C.] [ C.] [ C.] PBS 70.2 70.2 70.2 70.2 70.2 70.2 70.2 70.2 trehalose 73.6 73.5 74.2 74.2 73.5 73.0 72.6 73.1 chitosan 73.6 73.7 73.4 73.1 73.3 74.0 75.2 75.4 SPS 73.1 72.9 72.8 73.9 72.8 72.4 72.1 73.0 SPS + n.d. 72.8 73.1 n.d. 74.0 74.4 74.8 75.8 trehalose SPS + 73.5 73.6 73.3 74.1 75.5 78.1 n.d. 78.9 chitosan SPS + 73.3 73.1 73.2 73.8 75.7 77.6 80.4 81.5 trehalose + chitosan

TABLE-US-00004 TABLE 3 Overview of formulation variants. Formulations for the in vivo study are marked with an asterisk. SPS HA further Pandemrix (mg/ (g/ matrix components/ formulations mL) mL) (mg/mL) variations irradiation Original 15 Pandemrix* SD M 15 Mannitol, No 160 Yes* 25 kGy Yes 40 kGy SD T 15 Trehalose, No 160 Yes 25 kGy Yes 40 kGy SD SPS80 M 80 15 Mannitol, No 160 SD SPS80 T 80 15 Trehalose, No 160 Yes 25 kGy Yes 40 kGy SD SPS80 T80 80 15 Trehalose, No 80 SD SPS40 T80 40 15 Trehalose, No 80 Yes 25 kGy Yes 40 kGy SD SPSv1 T 80 15 Trehalose, Variation of No 160 SPS (no Yes 25 kGy hygroscopic Yes 40 kGy amino acids) SD SPS T 80 15 Trehalose, 2 mg/ml GA + No* final 160 2 mg/ml Yes* 25 kGy Chitosan-HCl

TABLE-US-00005 TABLE 4 Spray-drying parameters used. Parameters Values Two fluid nozzle 1.5 mm inner diameter Inlet air temperature 120 ( C.) Aspirator air flow 35 (m.sup.3/h) = 100% Flow rate 5-6 (ml/min) Spray flow rate 470 L/h Outlet air temperature 50-55 ( C.)

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